The streptokinase is a polypeptide of 414 amino acids residues. This is an extracellular protein produced by various strains of beta haemolytic streptococci, with molecular weight about 47,000 dalton and is a potent activator of the fibrinolytic enzyme system in humans (Tillet, W. S. and Garner, R. L. (1933) Exp. Med. 58, 485-502; Tillet, W. S.; Edwards, E. D. and Garner, R. L (1934) J. Clin. Invest 13, 47-78).
Unlike other plasminogen activators, streptokinase does not possess the intrinsic protease activity necessary to activate plasminogen to plasmin. Streptokinase activates plasminogen by the formation of 1:1 molar complex of streptokinase-plasminogen, which serves as the activator of free plasminogen to form plasmin (Schick. L. A. and Castellino, F. J. (1974) Biochem. Biophys. Res. Commun. 57, 47-54).
Streptokinase, urokinase and tissue-type plasminogen activator are at present used as thrombolytic agents in the treatment of disorders which collectively represent one of the greatest causes of death in the world, such as myocardial infarct, pulmonary thromboembolism, surgical complications and other cases of thrombosis.
The streptokinase is a bacterial protein and therefore, antigenic in humans. Antibodies to streptokinase are found in most individuals as a result of recurrent streptococcal infection (Tillet, W. S. and Garner. R. L. (1934) J. Clin. Invest. 13. 47-78). These antibodies are harmful for the use of streptokinase as thrombolytic, because high antibodies titers might neutralize streptokinase activity preventing effective thrombolysis (Urdahl. K. B.; Mathews. J. D.; Currie, B. (1996) Australian and New Zealand J. Med. 26, 49-53:; Sponl, F. and Kaiser. R. (1974) Thromb. Diath. Haemorrh.32. 608). Patients are also immunized with streptokinase as a result of thrombolytic therapy and anti-streptokinase antibody titers exponentially rise post-treatment. These high anti-streptokinase antibody titers could neutralize a standard dose of streptokinase if it is administered a second time in therapy (Rao, A. K.; Pran, C.; Berke, A.; Jaffe, A.; Ockene, L.; Schreiber, T. L.; Bell, W. R.; Knaterund, G.; Robertson, T. L. and Terrin, Ml.L. (1988) 3. Am. Coll. Cardiol. 11,1). One of the most common side effects of streptokinase therapy are allergic reactions, which have been noted in up to 15% of treated patients (McGrath, K. G.; Zeffren, B.; Alexander, J.; Kaplan, K. and Patterson, R. (1985) J. Allergy Clin. Immunol. 76, 453; Sorber, W. A. and Herbst, V. (1988) Cutis 42, 57; Davies, K. A.; Mathieson, P.; Winearis, C. G.; Rees, A. J.; and Walport, M. J. (1990) Clin.Exp.Immunol.80, 83; Schweitzer, D. H.; Van der Wall, E. E.; Bosker, H. A.; Scheffer, E. and Macfarlane, J. D. (1991) Cardiology 78, 68; Bruserund, O. L.; Sollid, L. and Foyn-Jorgensen, P. (1986) J. Clin. Lab. Immun. 20, 69-74). The streptokinase also induces a strong cellular immune response (Bruserund, O. (1990) APMIS 98, 1077-1084; Bruserund, O.; Elsayed, S. and Pawelec, G. (1992) Mol. Immunol. 29,1097-1104; Youkeles, L. H.; Soliman, M. Y. and Rosenstreich, D. L. (1991) J. Allergy Clin. Immunol. 88, 166-171; Randall, K.; Gelfond, D. H.; Stoffel, S.; Scharf, S.; Higuchi,R.; Hom, G. T.; Mullis, K. B. and Erlich, H. A. (1988) Science 239, 487-491).
The widespread use of streptokinase in humans makes its antigenicity an important clinical problem.
Despite the rich clinical information about on the immunogenicity of streptokinase, little is known about the structural basis for its antigenicity. There is no X ray crystallographic data on the structure of streptokinase and it is not known whether certain regions of the molecule are more immunogenic than others, nor have there been studies of the molecular mechanisms responsible for antibody-mediated neutralization of streptokinase activity. Previous reports have shown different antigenic regions in streptokinase mapped with murine anti-streptokinase monoclonal antibodies, soluble recombinant streptokinase fragment and anti streptokinase antibodies from human sera from patients treated with streptokinase (Reed, G. L.; Kussie, P. and Parhami-Seren, B. (1993) J. Immunol. 150, 4407-4415; Parhami-Seren, B.; Lynch, M.; White, H. D. and Reed, G. L. (1995) Mol. Immunol. 32, 717-724; Parhami-Seren, B.; Keel, T. and Reed, G. L. (1996) Hybridoma 15, 169-176; Gonzalezgronow, M.; Enghild, J. J.;
Pizzo, S. V. (1993) Biochimica et Biophysica Acta 1180,283-288; U.S. Pat. No. 5,240,845).
The object of the present invention is to achieve streptokinase mutants from modifications of skc-2 gene previously described (European Patent No. EP 0 489 201 B1; Estrada et al (1992) Biotechnology 10, 1138-1142) and coding for streptokinase SKC-2 (Heberkinase(copyright), Heber Biotec SA, Havana, Cuba), such that the obtained mutants conserve their capacity for plasminogen activator complex formation and having reduced antigenicity that could constitute a preferred alternatives to native streptokinase for thrombolytic therapy. Heberkinase(copyright) contains a recombinant SKC-2 obtained after the expression of the skc-2 gene in E. coli (European Patent No. EP 0 489 201 B1; Estrada et al (1992) Biotechnology 10, 1138-1142).